The present invention relates to complex compounds and methods for using such complex compounds. The compounds of the invention are preferably used in methods for targeting cellular receptors that facilitate endocytic processes. The present invention takes advantage of this receptor targeting to enhance the intracellular uptake of biologically active compounds for therapeutic purposes.
The use of synthetic, short, single stranded oligonucleotide sequences to inhibit gene expression has evolved to the clinical stage in humans. It has been demonstrated that incorporation of chemically modified nucleoside monomers into oligonucleotides can produce antisense sequences which can form more stable duplexes and can have high selectivity towards RNA (Frier et al., Nucleic Acids Research, 1997, 25, 4429-4443). Two modifications that have routinely given high binding affinity together with high nuclease resistance are phosphorothioates and methylphosphonates.
There are a number of desirable properties such as specificity, affinity and nuclease resistance that oligonucleotides should possess in order to elicit good antisense activity. The ability to selectively target and be taken up by diseased cells is another important property that is desirable in therapeutic oligonucleotides. Natural oligonucleotides are polyanionic and are known to penetrate cells at very low concentrations. Neutral oligonucleotides, such as the methylphosphonates, are taken up by cells at much higher concentrations. Although the processes by which antisense oligonucleotides enter the cell membrane are not well understood, there is substantial evidence for distinct mechanisms of cell entry based on the electronic character of the antisense sequence.
Delivery of an antisense oligonucleotide to a specific, diseased cell is a very important area of active research. The majority of projected antisense therapies are for viral infections, inflammatory and genetic disorders, cardiovascular and autoimmune diseases and significantly, cancer. For example, in conventional chemotherapy, neoplasticity and virus-related infections are treated with high drug concentrations, leading to overall high systemic toxicity. This method of treatment does not distinguish between diseased cells and healthy ones.
In the treatment of cancers, the ability of antisense agents to down-regulate or inhibit the expression of oncogenes involved in tumor-transforming cells has been well documented in culture and animal models. For example, antisense inhibition of various expressed oncogenes has been demonstrated in mononuclear cells (Tortora et al., Proc. Natl. Acad. Sci., 1990, 87, 705), in T-cells, in endothelial cells (Miller et al., P.O.P., Biochimie, 1985, 67, 769), in monocytes (Birchenall-Roberts et al., Suppl. 1989, 13 (P.t. C), 18), in reticulocytes (Jaskulski, et al., Science 1988, 240, 1544)and in many other cell types, as generally set forth in Table 1.
Virally infected cell cultures and studies in animal models have demonstrated the great promise of antisense and other oligonucleotide therapeutic agents. Exemplary targets from such therapy include eukaryotic cells infected by human immunodeficiency viruses(Matsukura et al., Proc. Natl. Acad. Sci., 1987, 84, 7706; Agrawal et al., Proc. Natl. Acad. Sci., 1988, 85, 7079), by herpes simplex viruses (Smith et al., P.O.P., Proc. Natl. Acad. Sci., 1986, 83, 2787), by influenza viruses (Zerial et al., Nucleic Acids Res., 1987, 15, 9909) and by the human cytomegalovirus (Azad et al., Antimicrob. Agents Chemother., 1993, 37, 1945). Many other therapeutic targets also are amenable to such therapeutic protocols.
The use of non-targeted drugs, to treat disease routinely causes undesirable interactions with non-diseased cells (Sidi et al., Br. J. Haematol., 1985, 61, 125; Scharenberg et al., J. Immunol., 1988, 28, 87; Vickers et al., Nucleic Acids Res., 1991, 19, 3359; Ecker et al., Nucleic Acids Res., 1993, 21, 1853). One example of this effect is seen with the administration of antisense oligonucleotide in hematopoietic cell cultures that exhibit non-specific toxicity due to degradative by-products.
Other research efforts suggest that antisense oligonucleotides possess more side effects in both in vitro and in vivo animal models. For example, non-complementary DNA sequences have been shown to interfere with cell proliferation and viral replication events through unknown mechanisms of action (Kitajima ibid). This reinforces the desirability of oligonucleotides that are specifically targeted to diseased cells.
When phosphodiester oligonucleotides are administered to cell cultures, a concentration of typically about 1 mmol is required to see antisense effects. This is expected since local endonucleases and exonucleases cleave these strands effectively and only 1-2% of the total oligonucleotide concentration becomes cell-associated (Wickstorm et al., Proc. Natl. Acad. Sci. 1988, 85, 1028; Wu-Pong et al., Pharm. Res. 1992, 9, 1010). If chemically modified oligonucleotides, such as the phosphorothioates or methylphosphonates are used, the observed antisense effects are anywhere between 1 and 100 xcexcM. This observed activity is primarily due to the relatively slow cellular uptake of oligonucleotides. There is evidence which suggests that a 80 kiloDalton (kDa) membrane receptor mediates the endocytic uptake of natural and phosphorothioate oligonucleotides in certain type of cells. Other data question the existence of such a link between receptor-mediated oligonucleotide uptake and internalization of oligonucleotides. For example, inhibitors of receptor-mediated endocytosis have no effect on the amount of oligonucleotide internalized in Rauscher cells (Wu-Pong et al., Pharm. Res., 1992, 9, 1010). For uncharged methylphosphonates, it was previously believed that internalization of such agents occurred by passive diffusion (Miller et al., Biochemistry 1981, 20, 1874). These findings were disproved by studies showing that methylphosphonates take up to 4 days to cross phospholipid bilayers, which correlates well with the fate of internalization of natural oligonucleotides (Akhtar et al., Nucleic Acids Res., 1991, 19, 5551).
Increased cellular uptake of antisense oligonucleotides by adsorptive endocytosis can be obtained by liposome encapsulation. In one study, researchers showed that a 21-mer complementary to the 3xe2x80x2-tat splice acceptor of the HIV-1 was able to markedly decrease the expression of a p24 protein while encapsulated into a liposome containing diastearoylphosphatidylethanol-amine (Sullivan et al., Antisense Res. Devel., 1992, 2, 187). Many other examples have been reported, including pH-sensitive liposomes (Huang et al., Methods Enzymol., 1987, 149, 88) which are well detailed in several good review articles (Felgner et al., Adv. Drug Deliv. Rev., 1990, 5, 163 and Farhood et al., N.Y. Acad. Sci., 1994, 716, 23). When phosphorothioate oligonucleotides, that are complementary to the methionine initiation codon of human intracellular adhesion molecule-1, were encapsulated, a 1000-fold increase of antisense potency was seen relative to the non-encapsulated phosphorothioate oligonucleotide (Bennett et al., Mol. Pharmacol., 1992, 41, 1023). The oligonucleotide delivery systems are good for in vitro cell systems, but have not been shown to be widely applicable to in vivo studies, due to rapid liposome destabilization and non-specific uptake by liver and spleen cells.
Other, non-specific oligonucleotide uptake enhancements attend attaching hydrophobic cholesterol (Letsinger et al., Proc. Natl. Acad. Sci., 1989, 86, 6553) type or phospholipid type molecules (Shea et al., Nucleic Acids Res., 1990, 18, 3777) to the oligonucleotides. It has been shown that the coupling of a single cholesterol moiety to an antisense oligonucleotide increases cellular uptake by 15-fold (Boutorin et al., FEBS Lett., 1989, 254, 129). When the cationic polymeric drug carrier poly(L-lysine) is conjugated to oligonucleotide sequences, a marked increase of non-specific oligonucleotide cellular uptake occurs (Lemaitre et al., Proc. Natl. Acad. Sci., 1987, 84, 648; Leonetti et al., Gene 1988, 72, 323; Stevenson et al., J. Gen. Virol., 1989, 70, 2673). This cationic polymer has been used to deliver several types of drugs with cellular uptake mediated by an endocytic-type mechanism. However, the high molecular weight polylysine is cytotoxic even at low concentrations.
Cell surface receptors are good candidates to serve as selective drug targets. The presence of specific receptors implies that natural endogenous ligands are also present. It is the complexation of the ligand with the appropriate receptor that elicits a cascade of cellular events leading to a desired function. An oligonucleotide drug linked to such an endogenous ligand or a synthetic ligand of equal affinity towards the receptor in question, is considered xe2x80x9ctargetedxe2x80x9d to the receptor.
The potential of carbohydrate drug targeting has become increasingly apparent (Shen et al., N.Y. Acad. Sci., 1987, 507, 272; Monsigny et al., N.Y. Acad. Sci., 1988, 551, 399; Karlsson et al., TIPS 1991, 12, 265) as an alternate method for site-specific drug delivery. Complex carbohydrates are involved in many cellular recognition processes such as adhesion between cells, adhesion of cells to the extracellular matrix, and specific recognition of cells (Ovarian egg with sperm) by one another (Yamada, K. M., Annu., Rev. Biochem., 1983, 52, 761; Edelman, G. M., Annu. Rev. Biochem., 1985, 54, 135; Hook et al., Annu. Rev. Biochem., 1984, 53, 847; Florman, H. M., Cell, 1985, 41, 313. It is also known that the concentrations of various glycosylated proteins that circulate in the blood are constantly regulated by cells in various tissues. Nature controls and regulates such diverse functions with the aid of specific proteins appearing at the surface of various cells, which have the ability to decode the information found in complex carbohydrate structures. These proteins are collectively called lectins and act as receptors for carbohydrates (Goldstein et al., Nature, 1980, 285, 66). Many endogenous lectins are expressed at the surface of normal and malignant cells and are involved in many poorly understood biological processes.
The structural information obtained from a large number of mammalian lectins has led to their classification into several families.
The C-lectins or calcium-dependent lectins possess carbohydrate recognition domains (CRDs) of the 115-134 amino acids which contain 18 highly conserved and 14 invariant residues (Drickamer, K., J. Biol. Chem., 1988, 263, 9557; Drickamer, K., Curr. Opin. Struc. Biol., 1993, 3, 393; Drickamer, K., Biochemical Society Transactions, 1993, 21, 456). The C-lectins are interesting from a pharmacological point of view since they recognize specific carbohydrates and immediately endocytose the receptor-bound glycoprotein complex via coated pits and vesicles.
These vesicles which are also referred to as endosomes, bring the receptor-glycoprotein complex to other cellular compartments, called the lysozomes, where protein degradation occurs (Breitfeld et al., Int. Rev. Cytol., 1985, 97, 4795). The range of C-type lectin carbohydrate specificity differs form cell to cell and from tissue to tissue.
The first membrane lectin was characterized on hepatocyte liver cells (Van Den Hamer et al., J. Mol. Biol., 1970, 245, 4397). The hepatic asialoglycoprotein receptor (ASGP-R) was isolated by Ashwell and Harford (Ashwell, G.; Herford, J., Ann. Rev. Biochem. 1982, 51, 531; Schwartz, A. L., CRC Crit. Rev. Biochem., 1984, 16, 207). These lectins internalized efficiently and cleared plasma levels from ceruloplasmin which contained abnormally truncated N-oligosaccharides lacking the terminal sialic acid residues. Other artificial molecules which have terminal galactose or N-acetylgalactosamine residues have been found to bind with high affinity to this lectin. This unique specificity between the exposed galactose units and the ASGP-R suggested the design and testing of glycotargeting systems and the use of lectins as specific drug delivery targets.
Many other cell lines, some summarized in Table 2, have surface carbohydrate-type receptors that mediate uptake of various ligands (Drickamer, K., Cell 1991, 67, 1029). Immune cells like monocytes and macrophages possess a number of surface glycoproteins that enable them to interact with invading micro-organisms (Gordon et al., J. Cell Sci. Suppl., 1988, 9, 1). Drugs need to be carried to target cells via a carrier or high affinity ligand which is attached to the drug. The different carriers for glycotargeting can be glycoproteins or neoglycoproteins, (glycopeptides or neoglycopeptides) and as glycosylated polymers.
The in vitro glycotargeting principle is relatively simple, but its in vivo applicability is difficult. Synthetic efforts have generated liposomes (also referred as immunoliposomes) and polylysine carriers, in which antibodies and some carbohydrate conjugate ligands have been covalently attached on the outer bilayer. For example, when natural oligonucleotides complementary to the translation initiation region of VSV N protein mRNA were encapsulated with liposomes whose outer membrane had several H2K-specific antibodies to L929 cells, there was a marked decrease in viral replication only within L929 infected cells (Leonetti et al., Proc. Natl. Acad. Sci., 1990, 87, 2448). Receptor-mediated endocytic mechanisms have been exploited by attachment of cell-specific ligands and antibodies to polylysine polymers. For example, c-myb antisense oligonucleotides conjugated with polylysine-folic acid (Citro et al., Br. J. Cancer 1992, 69, 463) or polylysine-transferrin (Citro et al., Proc. Natl. Acad. Sci., 1992, 89, 7031) targets were found to better inhibit HL-60 leukemia cell line proliferation than oligonucleotides without conjugated carriers. Another promising polylysine-asialoorosomucoid carrier was conjugated with phosphorothioate oligonucleotides complementary to the polyadenylation signal of Hepatitis B virus (Wu, G. Y.; Wu, C. H., J. Biol. Chem., 1992, 267, 12436).
Methods have been previously developed that utilize conjugates to enhance transmembrane transport of exogenous molecules. Ligands that have been used include biotin, biotin analogs, other biotin receptor-binding ligands, folic acid, folic acid analogs, and other folate receptor-binding ligands. These materials and methods are disclosed in U.S. Pat. No. 5,108,921, issued Apr. 28, 1992, entitled xe2x80x9cMethod for Enhanced Transmembrane Transport of Exogenous Moleculesxe2x80x9d, and U.S. Pat. No. 5,416,016, issued May 16, 1995, entitled xe2x80x9cMethod for Enhancing Transmembrane Transport of Exogenous Moleculesxe2x80x9d, the disclosures of which are herein incorporated by reference.
These immunoliposomes and antibody-polymer targeting exhibited no in vivo activity. With similar drawbacks as their non-specific counterparts, the immunoliposome-drug complexes are mostly immunogenic and are phagocytosed and eventually destroyed in the lysosome compartments of liver and spleen cells. As for the antibody-polylysine-drug complexes, they have shown substantial in vitro cytotoxic activity (Morgan et al., J. Cell. Sci., 1988, 91, 231). Other carriers are glycoproteins. On such large structures, a few drug molecules can be attached. The glycoprotein-drug complexes can subsequently be desilylated, either chemically or enzymatically, to expose terminal galactose residues.
Glycoproteins and neoglycoproteins are recognized by lectins such as the ASGP-R. Glycoproteins having a high degree of glycosylation heterogeneity are recognized by many other lectins making target specificity difficult (Spellman, N. W., Anal. Chem., 1990, 62, 1714). Neoglycoproteins having a high degree of homogeneity exhibit a higher degree of specificity for lectins especially the ASGP-R. Many experimental procedures which are used to couple sugars to proteins have been reviewed by Michael Brinkley (Brinkley, M., Bioconjugate Chem., 1992, 3, 2). These neoglycoproteins may mimic the geometric organization of the carbohydrate groups as in the native glycoprotein and should have predictable lectin affinities. Successful in vitro delivery of AZT-monophosphate, covalently attached to a human serum albumin containing several mannose residues was achieved in human T4 lymphocytes (Molema et al., Biochem. Pharmacol., 1990, 40, 2603).
Examples of antisense oligonucleotide-neoglycoprotein complexes have been previously reported (Bonfils et al., Nucleic Acids Res., 1992, 20, 4621). The authors mannosylated bovine serum albumin and attached, covalently from the 3xe2x80x2-end, a natural oligonucleotide sequence. The oligonucleotide-neoglycoprotein conjugate was internalized by mouse macrophages in 20-fold excess over the free oligonucleotide. Biotinylated oligonucleotides, were also disclosed which were non-covalently associated with mannosylated streptavidin (Bonfils et al., Bioconjugate Chem., 1992, 3, 277). Such complexes were also better internalized by macrophages. Other successful examples consisted of antisense oligonucleotides which were non-covalently associated with asialoglycoprotein-polylysine conjugates. Such oligonucleotide conjugates were found to internalize more efficiently into hepatocytes (Bunnel et al., Somatic Cell Molecular Genetics, 1992, 18, 559; Reinis et al., J. Virol. Meth., 1993, 42, 99) and into hepatitis B infected HepG2 cells (Wu, G. Y.; Wu, C. H., J. Biol. Chem., 1992, 267, 12436).
Polymeric materials have been assessed as drug carriers and three of them, dextrans, polyethyleneglycol (PEG) and N-(2-hydroxypropyl)methacrylamide (HMPA) co-polymers, have been successfully applied in vivo (Duncan, R., Anticancer Drugs, 1992, 3, 175). This research has been focused mainly at treatments for cancer and as a requisite the size of the compounds are between 30-50 kDa to avoid renal excretion (Seymour, L. W., Crit. Rev. Ther. Drug Carrier Syst., 1992, 9, 135).
In order to examine the chemistry and related methodologies involving the preparation of glycoprotein-drug and neoglycoprotein-drug glycoconjugates, certain groups have investigated the use of simpler high affinity ligands for specific drug delivery. Initially, several sugars were attached to small peptides in an attempt to obtain mimics of multivalent N-linked oligosaccharides (Lee, R. T., Lee, Y. C., Glycoconjugate J., 1987, 4, 317; Plank et al., Biconjugate Chem., 1992, 3, 533; Haensler et al., Bioconjugate Chem., 1993, 4, 85).
Other groups investigated the use of sugar clusters lacking a protein backbone and eventually used low molecular weight N-linked oligosaccharides with a minimum carbohydrate population to bind with high affinity to lectins as the ASGP-R. Branched N-linked oligosaccharide-drug conjugates can be used instead of neoglycoprotein-drug complexes. The total synthesis of branched N-linked oligosaccharides is still a difficult task, however they could be obtained by enzymatic cleavage from protein backbones (Tamura et al., Anal. Biochem., 1994, 216, 335). This method requires expensive purifications and only generates low quantities of chemically defined complex oligosaccharides. The affinity of N-linked oligosaccharide clusters towards many lectins has been demonstrated and has helped researchers to locate different new mammalian lectins in animals (Chiu et al., J. Biol. Chem., 1994, 269, 16195).
One process of increasing the intracellular oligonucleotide concentration is via receptor-mediated endocytic mechanisms. This novel drug targeting concept has been demonstrated in vitro by several groups. Oligonucleotides have been attached to glycoproteins, neoglycoproteins and neoglycopolymers possessing a defined carbohydrate population which, in turn, are specifically recognized and internalized by membrane lectins. To the best of our knowledge in vivo applicability of oligonucleotide-carbohydrate conjugates has not been previously demonstrated.
It has also been shown in in vitro experiments that synthetic neoglycoproteins containing galactopyranosyl residues at non-reducing terminal positions are recognized by the ASGP-R with increasing affinity as the number of sugar residues per molecule is increased (Kawaguchi et al., J. Biol. Chem. 1981, 256, 2230).
As in apparent, there exists a need for an improved method of selective delivery of biologically active compounds such as antisense oligonucleotides to specific cells. This invention is directed to providing methods to effect such delivery.
The present invention provides complexes and methods for using such complexes. The complex forms are useful for enhancing the intracellular uptake of biologically active compounds (primary compounds). The complex compounds of the invention are prepared having the component parts shown below: 
wherein the primary moiety is a nucleotide, nucleoside, oligonucleotide or oligonucleoside; each of said linkers are, independently, bi- or trifunctional; said manifold is derivatized at a plurality of sites;each of said cell surface receptor ligands is a carbohydrate; and n is an integer from 2 to about 8.
Preferably, at least two cell surface receptors are individually linked by linker groups to a manifold compound which is further linked to a primary compound. The cell surface receptor ligands impart affinity to the complexes for cells having surface receptors that recognize the selected cell surface receptor ligands. This interaction is believed to trigger endocytosis of the complex, resulting in an increased uptake by the cell of the primary compound.
In one embodiment of the invention primary compounds are selected to be oligonucleotides or oligonucleosides. Attachment of oligonucleotides or oligonucleosides to a manifold compound, can be conveniently made at the 5xe2x80x2 or 3xe2x80x2 phosphate of the 5xe2x80x2 or 3xe2x80x2 terminal nucleotide or nucleoside of the oligonucleotide or oligonucleoside. Alternatively, the phosphate group can be introduced as part of the linker group attached to the manifold moiety. Such a coupling is made by selecting the terminus of the linker to be a hydroxyl group and converting it to a phosphoramidite. The phosphoramidite can then be reacted with an unblocked 2xe2x80x2, 3xe2x80x2 or 5xe2x80x2 hydroxyl group of an oligonucleotide or oligonucleoside.
Manifold species as used in the present invention can include a wide variety of compounds that have functional groups or sites that can be linked by linker groups to a primary compound together with a cell surface receptor ligand, or, preferably ligands. In one embodiment a polycyclic molecule may be selected as the manifold compound, as can be illustrated for cholic acid. In other embodiments a smaller, monocyclic manifold compound can be selected, such as phenyl or cyclohexyl.
Manifold compounds can also comprise branched chain aliphatic compounds that have the funtionalities available for linking. Also, combinatorial chemistry techniques are known to utilize numerous compounds that can be used as manifold compounds. Many combinatorial scaffolds are ammenable for use as manifold compounds by virtue of their multiple reactive sites, which can be subjected to various orthogonal protection schemes. Preferable reactive sites include hydroxyl groups, carboxylic acid groups, amino groups and thiol groups.
Linker groups that are preferred for use in the present invention can be selected for a variety of chemical reasons. If the primary compound is an oligonucleotide or oligonucleoside, a linker can be conveniently chosen having a secondary hydroxyl group and/or a primary hydroxyl group with an additional functionality such as an amino, hydroxyl, carboxylic acid, or thiol group. The additional functionality can be used to attach one end of the linker group to the manifold moiety by for example an amide linkage. The secondary and or primary hydroxyl groups can be used to prepare a DMT or DMT phosphoramidite as illustrated in the Example section. This enables the attachment to an oligonucleotide or oligonucleoside to a solid support or to the 2xe2x80x2, 3xe2x80x2 or 5xe2x80x2 position or a ribosyl group. This will allow variability of the placement of the conjugated manifold compound to the primary compound. In a preferred embodiment an oligonucleotide is prepared using standard automated solid support protocols as is well known in the art and the conjugated manifold compound is coupled as the last step to the 5xe2x80x2-O position of the completed oligonucleotide or oligonucleoside. Cleavage from the solid support will give the complex compound.
For use in antisense and similar methodologies, oligonucleotides and oligonucleosides of the invention preferably comprise from about 10 to about 30 subunits. It is more preferred that such oligonucleosides comprise from about 15 to about 25 subunits. As will be appreciated, a subunit is a base and sugar combination suitably bound to adjacent subunits through, for example, a phosphorous-containing (e.g., phosphodiester) linkage or some other linking moiety. The nucleosides need not be linked in any particular manner, so long as they are covalently bound. Exemplary linkages are those between the 3xe2x80x2- and 5xe2x80x2-positions or 2xe2x80x2- and 5xe2x80x2-positions of adjacent nucleosides. Exemplary linking moieties are disclosed in the following references: Beaucage, et al., Tetrahedron 1992, 48, 2223 and references cited therein; and U.S. patent applications: Ser. No. 703,619, filed May 21, 1991; Ser. No. 903,160, filed Jun. 24, 1992; Ser. No. 039,979, filed Mar. 20, 1993; Ser. No. 039,846, filed Mar. 30, 1993; and Ser. No. 040,933, filed Mar. 31, 1993. Each of the foregoing patent applications are assigned to the assignee of this invention. The disclosure of each is incorporated herein by reference.
In other embodiments of the invention, primary compounds comprise oligonucleotides or oligonucleosides attached through a linking moiety to the manifold moiety such as by a free 2xe2x80x2-, 3xe2x80x2-, or 5xe2x80x2-hydroxyl group. Such attachments are prepared by, for example, reacting nucleosides bearing at least one free 2xe2x80x2-, 3xe2x80x2-, or 5xe2x80x2-hydroxyl group under basic conditions with a linking moiety having a leaving group such as a terminal Lxe2x80x94(CH2)xe2x80x94 etc. function, where L is a leaving group. Displacement of the leaving group through nucleophilic attack of (here) an oxygen anion produces the desired derivative. Leaving groups according to the invention include but are not limited to halogen, alkylsulfonyl, substituted alkylsulfonyl, arylsulfonyl, substituted arylsulfonyl, hetercyclcosulfonyl or trichloroacetimidate. A more preferred group includes chloro, fluoro, bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate), trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl, and 2,4,6-trichlorophenyl, with bromo being preferred.
Suitably protected nucleosides can be assembled into an oligonucleosides according to many known techniques. See, e.g., Beaucage, et al., Tetrahedron 1992, 48, 2223.
A wide variety of linker groups are known in the art that will be useful in the attachment of primary compounds to manifold compounds. Many of these are also useful for the attachment of cell surface receptor ligands to the manifold compound. A review of many of the useful linker groups can be found in Antisense Research and Applications, S. T. Crooke and B. Lebleu, Eds., CRC Press, Boca Raton, Fla., 1993, p. 303-350. A disulfide linkage has been used to link the 3xe2x80x2 terminus of an oligonucleotide to a peptide (Corey, et al., Science 1987, 238, 1401; Zuckermann, et al., J. Am. Chem. Soc. 1988, 110, 1614; and Corey, et al., J. Am. Chem. Soc. 1989, 111, 8524). Nelson, et al., Nuc. Acids Res. 1989, 17, 7187 describe a linking reagent for attaching biotin to the 3xe2x80x2-terminus of an oligonucleotide. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commercially available from Clontech Laboratories (Palo Alto, Calif.) under the name 3xe2x80x2-Amine on. It is also commercially available under the name 3xe2x80x2-Amino-Modifier reagent from Glen Research Corporation (Sterling, Va.). This reagent was also utilized to link a peptide to an oligonucleotide as reported by Judy, et al., Tetrahedron Letters 1991, 32, 879. A similar commercial reagent (actually a series of such linkers having various lengths of polymethylene connectors) for linking to the 5xe2x80x2-terminus of an oligonucleotide is 5xe2x80x2-Amino-Modifier C6. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or similar ones were utilized by Krieg, et al., Antisense Research and Development 1991, 1, 161 to link fluorescein to the 5xe2x80x2-terminus of an oligonucleotide. Other compounds such as acridine have been attached to the 3xe2x80x2-terminal phosphate group of an oligonucleotide via a polymethylene linkage (Asseline, et al., Proc. Natl. Acad. Sci. USA 1984, 81, 3297).
Oligonucleotides have been prepared on solid support and then linked to a peptide via the 3xe2x80x2 hydroxyl group of the 3xe2x80x2 terminal nucleotide (Haralambidis, et al., Tetrahedron Letters 1987, 28, 5199). An Aminolink 2 (Applied Biosystems, Foster City, Calif.) has also been attached to the 5xe2x80x2 terminal phosphate of an oligonucleotide (Chollet, Nucleosides and Nucleotides 1990, 9, 957). This group also used the bifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) to link an interleukin protein to an oligonucleotide.
In another embodiment of the invention, linker moieties are used to attach manifold groups to the 5 position of a pyrimidine (Dreyer, et al., Proc. Natl. Acad. Sci. USA 1985, 82, 968). Fluorescein has been linked to an oligonucleotide in the this manner (Haralambidis, et al., Nucleic Acid Research 1987, 15, 4857) and biotin (PCT application PCT/US/02198). Fluorescein, biotin and pyrene were also linked in the same manner as reported by Telser, et al., J. Am. Chem. Soc. 1989, 111, 6966. A commercial reagent, Amino-Modifier-dT, from Glen Research Corporation (Sterling, Va.) can be utilized to introduce pyrimidine nucleotides bearing similar linking groups into oligonucleotides.
Cholic acid linked to EDTA for use in radioscintigraphic imaging studies was reported by Betebenner, et.al., Bioconjugate Chem. 1991, 2, 117.
In a preferred embodiment of the present invention, novel complex compounds are prepared having oligonucleotide conjugates that are useful for oligonucleotide antisense drug targeting of, for example, the carbohydrate recognition domains (CRD) found on the asiologlycoprotein-receptor (ASGP-R). These complex compounds were prepared according to the principles which govern the specificity of the {ligand-[ASGP-R]} complex. Simple carbohydrates and Glycoconjugates having only one linked saccharide moiety show a slight affinity for the receptor (Lee et al., Biol. Chem., 1983, 258, 199). For instance, glycoconjugates having monovalent ligands such as galactose, lactose or monoantennary galactosides (one carbohydrate group attached via a linkage to the scaffold) bind to this ASGP-R with a millimolar dissociation constant. When binary oligosaccharides (two carbohydrate groups each attached via a linkage to the scaffold) are used, the dissociation constants are in the micromolar range. This translates to a three order of magnitude higher affinity. When trinary oligosaccharides (three carbohydrate groups each attached via a linkage to the scaffold) are tested, the dissociation constants are in the nanomolar range.
Based on dissociation constants, e.g. higher for 3 carbohydrate groups, trinary oligosaccharides were preferably synthesized, each having three carbohydrate groups independently linked to a scaffold which was further linked to an oligonucleotide. The resulting low molecular weight oligonucleotide conjugates were easily amenable to automated DNA synthesis methodology. The oligonucleotide conjugates each consist of at least four distinct moieties, scaffold, carbohydrate attaching linker, oligonucleotide attaching linker, and carbohydrate.
Initially, cholic acid was chosen as a scaffold. Cholic acid was chosen because it is a natural product in mammalian systems, does not form a toxic metabolite and because it is commercially available at low cost. Another reason for choosing cholic acid was that this steroidal scaffold would be a good anchor for linked carbohydrates, separating the points of attachment and reducing any steric interference between them. Increasing the distance between points of attachment of the linked carbohydrates would increase the degree of freedom and reduce the length requirement of the linker to obtain high affinity with the receptor.
Galactose and lactose were initially chosen as carbohydrate moieties since they are recognized by the carbohydrate recognition domains (CRD) found on the asiologlycoprotein-receptor (ASGP-R).
Aminocaproate, derived from commercially available N-Fmoc-xcex5-aminocaproic acid, was chosen as the carbohydrate linker and its length was based on previously reported experimental evidence (Biessen et al., J. Med.Chem., 1995, 38, 1538). The previously reported results indicated that cluster galactosides between 10 and 20 xc3x85 in length were high affinity substrates for the hepatic ASGRP-R. The length of the linking group was initially chosen to be eight atoms long because in conjunction with the larger area scaffold being used the result may be better positioning of the carbohydrate components towards the CRD""s of ASGP-R.
After the cholic acid scaffold has been linked to each of the carbohydrates and a linker group is deblocked and ready for attachment an oligonucleotide is coupled. The preferred method of coupling of the conjugate to an oligonucleotide is to perform the coupling while the full length oligonucleotide is bound to solid support. The conjugate is coupled to the oligonucleotide followed by cleavage of the final product from the solid support. This cleavage step also removes acetyl protecting groups present on any hydroxyl groups that were previously protected especially on any saccharide moieties.
The oligonucleotide analog is tested for affinity towards the ASGP-R expressed on several cells. The binding of the oligonucleotide conjugate to the ASGP-R should initiate the internalization process and increase the intracellular concentration of the selected oligonucleotide.
It will be appreciated that modified nucleotide moieties may also be useful in connection with embodiments of this invention. Thus, a wide variety of chemical modifications may be employed throughout the nucleic acids. Thus modifications of pyrimidine or purine bases, substitutions at the 2xe2x80x2 position, alteration of inter-nucleoside linkages, carbohydrate ring substitutions and positional variations may all be employed.
Teachings regarding the synthesis of modified oligonucleotides may be found in the following U.S. patents or pending patent applications, each of which is commonly assigned with this application: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023, drawn to backbone modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having xcex2-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 2xe2x80x2-O-alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, issued Jun. 29, 1993, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4xe2x80x2-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone modified oligonucleotide analogs; and U.S. patent application Ser. No. 08/383,666, filed Feb. 3, 1995, and U.S. Pat. No. 5,459,255, drawn to, inter alia, methods of synthesizing 2xe2x80x2-fluoro-oligonucleotides.
It is to be understood that all modifications to nucleotides, nucleosides at oligonomers thereof are encompassed within their respective definitions.
Antisense oligonucleotides are conversely synthesized using automated DNA synthetic methodology. Therefore, small glycotargeting systems, which can be incorporated into the last cycle of an automated DNA synthesis, are preferred.